Column temperature = 30 °C; flow rate = 1.0 ml/min; injected volume =20 μl; detection wavelength (λ)=254 nm were used. The mobile phase was a gradient elution program with a binary mixture of acetonitrile (solvent A) and H2O (solvent B), as Table 1:
Each HPLC run took about 25 min and afterwards the column was conditioned again with a mixture of 65% solvent A and 35% solvent B.